Anomalous diffusion of fluorescent probes inside living cell nuclei investigated by spatially-resolved fluorescence correlation spectroscopy

We have investigated spatial variations of the diffusion behavior of the gr een fluorescent protein mutant EGFP (F64L/S65T) and of the EGFP-beta-galact osidase fusion protein in living cells with fluorescence correlation spectr oscopy. Our fluorescence correlation spectroscopy device, in connection wit h a precision x-y translation stage, provides submicron spatial resolution and a detection volume smaller than a femtoliter. The fluorescence fluctuat ions in cell lines expressing EGFP are caused by molecular diffusion as wel l as a possible internal and a pH-dependent external protonation process of the EGFP chromophore. The latter processes result in two apparent nonfluor escent states that have to be taken into account when evaluating the fluore scence correlation spectroscopy data. The diffusional contribution deviates from ideal behavior and depends on the position in the cell. The fluoresce nce correlation spectroscopy data can either be evaluated as a two componen t model with one fraction of the molecules undergoing free Brownian motion with a diffusion coefficient approximately five times smaller than in aqueo us solution, and another fraction diffusing one or two orders of magnitude slower. This latter component is especially noticeable in the nuclei. Alter natively, we can fit the data to an anomalous diffusion model where the tim e dependence of the diffusion serves as a measure for the degree of obstruc tion, which is large especially in nuclei. Possible mechanisms for this lon g tail behavior include corralling, immobile obstacles, and binding with a broad distribution of binding affinities. The results are consistent with r ecent numerical models of the chromosome territory structure in the cell nu cleus. (C) 2000 Academic Press.